Measuring chip for surface plasmon resonance biosensor and method for producing the same

ABSTRACT

An objective of the present invention is to provide a measuring chip for a surface plasmon resonance sensor that can detect a small amount of target substances in high sensitivity. The present invention provides a measuring chip for a surface plasmon resonance sensor comprising a metal layer, one or more plasma polymerization layers formed on said metal layer, and a biologically active substance immobilized on the surface of said plasma polymerization layer.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to a surface plasmon resonancebiosensor, specifically, a measuring chip for the same and a method forproducing the measurement chip.

[0003] 2. Background Art

[0004] A number of methods using immunological reactions are used inclinical tests for detecting target substances. Conventional methods areknown to be intricate and require labeling substances. Thus,immunological sensors using a surface plasmon resonance biosensor (SPR)is being used, in which no labeling substance is required and a ligandcan be detected with high sensitivity. This surface plasmon resonancebiosensor is based on the phenomenon that the intensity of amonochromatic light reflected from the interface between an opticallytransparent substance such as glass and a metal thin-film layer isdependent on the refractive index of a sample placed on the reflectingside of the metal. Accordingly, a sample can be analyzed by measuringthe intensity of the reflected monochromatic light.

[0005] An optical part of a measuring cell for this surface plasmonresonance (surface plasmon resonance biosensor) has a structure shown inFIG. 2. Namely, porous material 5 is formed on metal thin-film 2 formedon glass substrate 1, and physiologically active substance 4, such as anenzyme or antibody, is retained or immobilized on the surface or insideof porous material 5. Examples of porous material 5 to be used includeweaved, knitted or non-woven cloths made of synthetic fibers, naturalfibers, inorganic fibers or the like, and porous inorganic or organicmaterials (see Japanese Patent Laid-open No. 164195/1991). Furthermore,carboxymethyl dextran is used as a porous material in a commercialproduct (BIAcore 2000, Pharmacia Biosensor).

[0006] However, physiologically active substance 4 just exists on thesurface of porous material 5 and interacts with target substances.

[0007] LB (Langmuir-Blodgett) method is occasionally used to immobilizephysiologically active substance 4 on metal thin-film 2 (see JapanesePatent Laid-open No. 288672/1993). However, this method has adisadvantage in that LB membrane binds poorly to a metal thin-film andpeels off together with the physiologically active substance.

[0008] Furthermore, Japanese Patent Laid-open No. 264843/1997 disclosesmeasuring cells for a surface plasmon resonance biosensor.

SUMMARY OF THE INVENTION

[0009] The present inventors have now found that sensitivity of ameasuring chip for a surface plasmon resonance sensor is extremelyimproved when only a small amount of a physiologically active substanceis immobilized on a specific plasma polymerization layer.

[0010] An objective of the present invention is to provide a measuringchip for a surface plasmon resonance sensor that can detect a smallamount of target substances in high sensitivity.

[0011] Another objective of the present invention is to provide ameasuring cell for a surface plasmon resonance sensor that can detect asmall amount of target substances in high sensitivity.

[0012] Further objective of the present invention is to provide a methodfor producing said measuring chip.

[0013] The present invention provides a measuring chip for a surfaceplasmon resonance sensor comprising a metal layer and one or more plasmapolymerization layers formed on said metal layer.

[0014] The present invention also provides a measuring chip for asurface plasmon resonance sensor comprising a metal layer, one or moreplasma polymerization layers formed on said metal layer, and abiologically active substance immobilized on the surface of said plasmapolymerization layer.

[0015] The present invention also provides a measuring cell for asurface plasmon resonance sensor comprising said measuring chip.

[0016] The present invention also provides a method for producing ameasuring chip for a surface plasmon resonance sensor comprising thesteps of forming a metal layer on an optically transparent substrate,forming one or more plasma polymerization layers on said metal layer,and then immobilizing a biologically active substance on the surface ofsaid plasma polymerization layer.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 is a schematic sectional view of one embodiment of themeasuring chip for a surface plasmon resonance sensor according to thepresent invention.

[0018]FIG. 2 is a schematic sectional view of an optical part of ameasuring chip for a conventional surface plasmon resonance biosensor.1: Transparent substrate; 2: Metal thin-film; 3: Plasma polymerizationlayer; 4: Physiologically active substance; 5: Porous material.

[0019] FIGS. 3 (a) and (b) each show a schematic sectional view of anoptical part of a measuring chip for a surface plasmon resonancebiosensor. (a) shows immobilization of an Fab fragment of an antibody.(b) shows immobilization of anF(ab′)_(2 fragment of an antibody. 1: Transparent substrate; 2: Metal thin-film; 3: Plasma polymerization layer; 4: Physiologically active substance.)

[0020]FIG. 4 is a schematic sectional view of an optical part of ameasuring chip for a surface plasmon resonance biosensor.

[0021]FIG. 5 illustrates a surface plasmon resonance biosensor. 7:Cartridge block; 8: Light source; 9: Detector; 10: Measuring chip; 71:Measuring cell; 72, 73: Flow routes; 80: Incident light; 90: Reflectinglight.

[0022]FIG. 6 illustrates a reflected light intensity curve before andafter plasma polymerization membrane formation.

[0023]FIG. 7 illustrates a schematic view showing the apparatus used inExample 1.

[0024]FIG. 8 shows the relationship between the concentration of thecomplementary DNA and RU in Example 1.

[0025]FIG. 9 shows the relationship between the concentration of thecomplementary DNA and RU in Example 2.

[0026]FIG. 10 shows the relationship between the concentration of thecomplementary DNA and RU in Example 3.

[0027]FIG. 11 shows the relationship between the concentration of thecomplementary DNA and RU in Example 4.

[0028]FIG. 12 shows the relationship between the concentration of theHSA antigen and RU in Example 5.

[0029]FIG. 13 shows the relationship between the concentration of theBSA antigen and RU in Example 6.

[0030]FIG. 14 shows the relationship between the concentration of thesugar and RU in Example 7.

[0031]FIG. 15 shows the relationship between the concentration of theBSA antigen and RU in Example 8.

[0032]FIG. 16 shows the relationship between the concentration of theBSA antigen and RU in Example 9.

[0033]FIG. 17 shows the relationship between the concentration of theBSA antigen and RU in Example 10.

[0034]FIG. 18 shows the relationship between the concentration of theBSA antigen and RU in Example 11.

[0035]FIG. 19 shows the relationship between the concentration of theHSA antigen and RU in Example 12.

[0036]FIG. 20 shows the relationship between the concentration of theHSA antigen and RU in Example 13.

[0037]FIG. 21 shows the relationship between the concentration of theHSA antigen and RU in Example 14.

[0038]FIG. 22 shows the relationship between the concentration of theHSA antigen and RU in Example 15.

[0039]FIG. 23 shows the relationship between the concentration of theHSA antigen and RU in Example 16.

[0040]FIG. 24 shows the relationship between the concentration of thecomplementary DNA and RU in Example 17.

[0041]FIG. 25 shows the relationship between the concentration of theHSA antigen and RU in Example 18.

[0042]FIG. 26 shows the relationship between the concentration ofskatole and RU in Example 19.

[0043]FIG. 27 shows the relationship between the concentration of theHSA antigen and RU in Example 20.

[0044]FIG. 28 shows the relationship between the concentration of theHSA antigen and RU in Example 21.

DETAILED DESCRIPTION OF THE INVENTION

[0045] The measuring chip for a surface plasmon resonance sensor(“measuring chip”) may have optically transparent substrate (transparentsubstrate) 1, metal thin-film 2 formed on transparent substrate 1,plasma polymerization layer 3 formed on metal thin-film 2, andphysiologically active substance 4 immobilized on the surface of plasmapolymerization layer 3 as shown in FIG. 1.

[0046] Transparent substrate 1 can be any substrate customarily used ina measuring chip for a surface plasmon resonance sensor. Generally,substrates made of materials that are transparent to a laser beam suchas glass can be used. The thickness of the substrate can be about 0.1 to5 mm.

[0047] Metal thin-film 2 is not particularly restricted, provided it caninduce surface plasmon resonance. Examples of the metal to be used formetal thin-film 2 include gold, silver and platinum. They can be usedalone or in combination. Furthermore, for better adhesion to transparentsubstrate 1, an auxiliary layer made of chrome or the like may be setbetween transparent substrate 1 and the layer made of gold, silver orthe like.

[0048] The thickness of metal thin-film 2 is preferably 100 to 2000angstroms, most preferably 100 to 500 angstroms. If the thicknessexceeds 3,000 angstroms, surface plasmon phenomena of the medium cannotbe sufficiently detected. Furthermore, when an auxiliary layer made ofchrome or the like is formed, the thickness of the auxiliary layer ispreferably 30 to 50 angstroms.

[0049] Plasma polymerization layer 3 can be formed by plasmapolymerization of a monomer material for three-dimensionalcross-linking. A monomer material to be used in the present inventioncan be any material that can immobilize a physiologically activesubstance by plasma polymerization.

[0050] Examples of a monomer material for a plasma polymerization layerinclude compounds of formula (I):

CH₃—(CH₂)n—NH₂ (wherein n is an integer from 1 to 6)  (I)

[0051] and compounds of formula (II):

NH₂—(CH₂)_(n)—NH₂ (wherein n is an integer from 1 to 6)  (II)

[0052] and compounds which comprise carbon (C), hydrogen (H) andnitrogen (N) and have double bonds or triple bonds, such asacetonitrile, vinylamine and pyridine.

[0053] Furthermore, when a cross-linking reagent or a condensationreagent is used as a linking layer, a compound further containing sulfur(S), oxygen (O) or silicon (Si) can be used as a monomer material.Generally, a compound appropriately containing any two or more elementsselected from carbon (C), hydrogen (H), nitrogen (N), sulfur (S), oxygen(O) and silicon (Si) can be used. In addition, a halogen gas or a raregas can be used as a monomer material.

[0054] In the present invention, a compound containing nitrogen can beused as a monomer material. Examples of the compound containing nitrogeninclude nitrogen N₂; ammonium; hydrazine; pyridine; compounds offormulae (I) and (II) such as ethylenediamine NH₂(CH₂)₂NH₂,hexamethylenediamine NH₂(CH₂)₆NH₂, n-propylamine CH₃(CH₂)₂NH₂ andmonoethylamine CH₃(CH₂)NH; compounds of formula (CH₃)₃(CH₂)_(n)N (n=0 to17) such as triethylamine (C₂H₅)₃N; compounds of formula(CH₃)₂(CH₂)_(n)NH (n=0 to 17) such as diethylamine (C₂H₅)₂NH; compoundsof formula CH₂═CH(CH₂)_(n)NH₂ (n=0 to 17) such as allylamineCH₂═CHCH₂NH₂; compounds of formula CH₃(CH₂)_(n)CN (n=0 to 17) such asacetonitrile CH₃CN; compounds of formula CH₃(CH₂)_(n)CN; propargylamineCHCCH₂NH₂; compounds of formula CHC (CH₂)_(n)NH₂; acrylamide; aniline;acrylonitrile; 1,2,4-triazole; and 5-amino-1H-tetrazole.

[0055] Further examples of of the compound containing nitrogen includethe following:

[0056] RaNRb₂:

[0057] Ra is H or CH₃(CH₂)_(n) (n=0 to 17),

[0058] and includes a group having a double bond or a triple bond orboth in the chain, and further a branched or cyclized group, and

[0059] Rb is H or CH₃(CH₂)_(n) (n=0 to 17),

[0060] and includes a group having a double bond or a triple bond orboth in the chain, and further a branched or cyclized group;

[0061] RaNRc:

[0062] Rc is H or CH₃(CH₂)_(n)CH (n=0 to 17), or CH₂,

[0063] and includes a group having a double bond or a triple bond orboth in the chain, and further a branched or cyclized group;

[0064] RdN:

[0065] Rd is CH₃(CH₂)_(n)C (n=0 to 17) or CH,

[0066] and includes a group having a double bond or a triple bond orboth in the chain, and further a branched or cyclized group;

[0067] ReNRfNRg₂:

[0068] Re is H or CH₃(CH₂)_(n) (n=0 to 17),

[0069] and includes a group having a double bond or a triple bond orboth in the chain, and further a branched or cyclized group,

[0070] Rf is (CH₂)_(n) (n=0 to 17),

[0071] and includes a group having a double bond or a triple bond orboth in the chain, and further a branched or cyclized group,

[0072] Rg is H or CH₃(CH₂)_(n) (n=0 to 17),

[0073] and includes a group having a double bond or a triple bond orboth in the chain, and further a branched or cyclized group;

[0074] RhNRiNRj

[0075] Rh is H or CH₃(CH₂)_(n) (n=0 to 17) or CH₃(CH₂)_(n) CH (n=0 to17) or CH)₂,

[0076] and includes a group having a double bond or a triple bond orboth in the chain, and further a branched or cyclized group,

[0077] Ri is (CH₂)_(n) (n=0 to 17) or CH (CH₂)_(n) CH (n=0 to 17),

[0078] and includes a group having a double bond a triple bond or bothin the chain, and further a branched or cyclized group,

[0079] Rj is H or CH₃(CH₂)_(n) CH (n=0 to 17) or CH₃(CH₂)_(n)CH (n=0 to17) or CH₂ or CH₃(CH₂)_(n) C (n=0 to 17) or CH,

[0080] and includes a group having a double bond or a triple bond orboth in the chain, and further a branched or cyclized group;

[0081] NRkN:

[0082] Rk is C(CH₂)_(n)C (n=0 to 17),

[0083] and includes a group having a double bond or a triple bond orboth in the chain, and further a branched or cyclized group.

[0084] In the present invention, a compound containing sulfur can beused as a monomer material. Examples of the compound containing sulfurinclude hydrogen sulfide; carbon disulfide thiophene; compounds offormula CH₃S(CH₂)_(n)CH₃ (n=0 to 17) such as dimethyl sulfide (CH₃)₂S;compounds of formula CH₃(CH₂)_(n)SS(CH₂)_(m)CH₃ (n=0 to 17, m=0 to 17)such as methyl disulfide CH₃SSCH₃; compounds of formula CH₃(CH₂)_(n)SH(n=0 to 17) such as ethanethiol CH₃CH₂SH; compounds of formulaSH(CH₂)_(n)SH (n=1 to 17) such as ethanedithiol SH(CH₂)₂SH;mercaptoethanol; and dithreitol.

[0085] Furthermore, compounds having one of or two or more of groupsincluding —COOH, —CHO, —SH, —NH₂, —OH, ═NH, CONH₂, —NCO, —CH═CH₂, ═C═Oand

[0086] can be used as a monomer material. Examples of such compoundsinclude cysteine, glutathione, formyl succinate, aminobenzoate,aminohexanate, mercaptobenzoate, and compounds having —C≡CCH₂OH.

[0087] In the present invention, a compound containing a halogen can beused as a monomer material. Examples of the compound containing ahalogen for a plasma polymerization layer include tetrafluoroethylene,chlorobenzene, hexachlorobenzene, hexafluorobenzene, and vinyl fluoride.

[0088] In the present invention, an organic metal compound can be usedas a monomer material. Examples of an organic metal compound for theplasma polymerization layer include an organic silicon compounds such astetramethylsilane, tetramethyldisiloxane, hexamethyldisiloxane,hexamethyldisilazane, hexamethylcyclotrisilazane,dimethylaminotrimethylsilane, trimethylvinylsilane, tetramethoxysilane,aminopropyltriethoxysilane, octadecyldiethoxymethylsilane,hexamethyldisilane and divinyltetramethyldisiloxane.

[0089] Compounds of formulae (I) and (II) having no double bond ortriple bond are preferably used because a layer is formed slowly so thatthe resulting layer is more homogeneous, compared with compounds havingdouble bonds or triple bonds.

[0090] The thickness of plasma polymerization layer 3 is preferably 100to 3000 angstroms, most preferably 500 to 1000 angstroms.

[0091] Plasma polymerization layer 3 can be formed by plasma treatmentto a resulting plasma layer with a polymeric or non-polymeric monomer.Examples of such non-polymeric monomer material include nitrogen,ammonium, hydrazine, hydrogensulfide, hydrogendisulfide, oxygen,hydrogen, water, halogen gas, and rare gas (e.g., argon, neon, helium,krypton, and xenon).

[0092] Furthermore, a mixture of various kinds of monomer materials canbe used as a monomer material. Plasma polymerization layer 3 can also beformed by lamination techniques and optionally using a mixture as amonomer material.

[0093] Plasma polymerization layer 3 of the present invention has thefollowing advantages:

[0094] 1) The layer is pinhole-free, amorphous, and dense.

[0095] 2) A thin homogeneous layer down to about 500 angstroms can bemade, which exhibits extremely little fluctuation in its refractiveindex.

[0096] 3) By changing the kind of plasma gas, not only a change in thethickness of the layer but also surface modification and surfaceimprovement, such as introduction of functional groups, and control ofthe density of the functional groups to be introduced can be attained.

[0097] 4) The layer can be formed in combination with semiconductortechniques since it is formed under dry conditions.

[0098] 5) The layer has excellent drug tolerance, heat tolerance,mechanical properties, and stability.

[0099] Furthermore, in the case of a sensor chip for SPR, in which ametal thin-film is essential, the metal thin-film and the plasmapolymerization layer can be formed in the same chamber. Thus, themanufacturing process can be simplified.

[0100] It is also advantageous to attain surface improvement, such asintroduction of a functional group, by further exposing a resultingplasma polymerization layer to plasma treatment with a non-polymeric orpolymeric monomer. The plasma polymerization treatment is intended toinclude a treatment with not only a non-polymeric monomer and aninactive monomer but also a polymeric monomer.

[0101] Physiologically active substance 4 to be immobilized is notparticularly limited, provided it reacts interacts with a targetsubstance to be measured. Examples of physiologically active substance 4include nucleic acids (e.g., DNA, RNA, and PNA); non-immune proteins(e.g., avidin (streptoavidin), biotin or a receptor);immunoglobulin-binding proteins (e.g., protein A, protein G and arheumatoid factor (RF)); sugar-binding proteins (e.g., lectin);sugar-recognizing sugar chains; fatty acids or fatty acid esters (e.g.,stearic acid, alachidic acid, behenic acid, ethyl stearate, ethylarachidate, and ethyl behanate); polypeptides or oligopeptides havingligand binding activity; immune proteins (e.g., an antibody); andenzyme.

[0102] When an antibody is used as physiologically active substance 4,Fc fragments of the antibody can be immobilized only on the surface ofplasma polymerization layer 3 and the antibody is formed in amonomolecular layer as shown in FIG. 1. However, since the sensitivityand the reaction rate decrease as Fab fragments of the antibody areseparated from plasma polymerization layer 3, Fab fragments (FIG. 3 (a))or F(ab′)₂ fragments (FIG. 3 (b)) can be immobilized directly on plasmapolymerization layer 3 as shown in FIG. 3 to improve the sensitivity andthe reaction rate.

[0103] The thickness of physiologically active substance 4 depends onthe size of the physiologically active substance itself, but ispreferably 100 to 3000 angstroms, most preferably 100 to 1000 angstroms.

[0104] In the present invention, the physiologically active substancecan be immobilized on the plasma polymerization layer through linkingagents.

[0105]FIG. 4 is a schematic illustration showing one embodiment of themeasuring chip according to the present invention. The measuring chiphas covalent bond layer 6 between plasma polymerization layer 3 andphysiologically active substance 4. Substance 4 is immobilized on plasmapolymerization layer 3 via covalent layer 6. The covalent bond can beformed with a cross-linking reagent or a condensation reagent.

[0106] The cross-linking reagent or a condensation reagent is notparticularly restricted, provided it can covalently and firmlyimmobilize substance 4. They can be used alone or in combination.

[0107] Examples of such cross-linking reagents include glutaraldehyde,periodic acid, N-succinimidyl-2-maleimidoacetic acid,N-succinimidyl-4-maleimidobutyric acid,N-succinimidyl-6-maleimidohexanic acid,N-succinimidyl-4-maleimidomethylcyclohexan-1-carboxylic acid,N-sulfosuccinimidyl-4-maleimidomethylcyclohexane-1-carboxylic acid,N-succinimidyl-4-maleimidomethylbanzoic acid,N-succinimidyl-3-maleimidobenzoic acid,N-sulfosuccinimidyl-3-maleimidobenzoic acid,N-succinimidyl-4-maleimidophenyl-4-butyric acid,N-sulfosuccinimidyl-4-maleimidophenyl-4-butyric acid,N,N′-oxydimethylene-dimaleimide, N,N′-o-phenylene-dimaleimide,N,N′-m-phenylene-dimaleimide, N,N′-p-phenylene-dimaleimide,N,N′-hexamethylene-dimaleimide, N-succinimidylmaleimidocarboxylic acid,N-succinimidyl-S-acetylmercaptoacetic acid,N-succinimidyl-3-(2-pyridyldithio)propionate, S-acetylmercaptosuccinicanhydride, methyl-3-(4′-dithiopyridyl)propionimidate,methyl-4-mercaptobutylimidate, methyl-3-mercaptopropionimidate,iminothiolene, o-carboxymethyl-hydroxylamine, azodiphenylpilmaleido,bis(sulfosuccinimidyl)sperate, 4,4′-diisothiocyano-2,2′-disulfonic acidstilbene, 4,4′-difluoro-3,3′-dinitrodiphenylsulfon,1,5-difluoro-2,4-dinitrobenzene, p-phenylenediisothiocyanate,dimethyladipimidate, dimethylpimelimidate, dimethylsuberimidate,p-azidophenacylbromide, p-azidophenylglyoxal,N-hydroxysuccinimidyl-4-azidobenzoate, 4-fluoro-3-nitrophenylazide,methyl-4-azidobenzoimidate, N-5-azido-2-nitrobenzoyloxysuccinimide,N-succinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate,1,4-benzoquinone, N-succinimidyl-3-(2′-pyridyldithio)propionate,N-(4-maleimidobutyloxy)sulfosuccinimide sodium salt,N-(6-maleimidocaproyloxy)sulfosuccinimide sodium salt,N-(8-maleimidocaproyloxy)sulfosuccinimide sodium salt,N-(11-maleimidoundecanoyloxy)sulfosuccinimide sodium salt,N-[2-(1-piperazinyl)ethyl]maleimide bichloric acid, bisdiazobenzidine,hexamethylenediisocyanate, toluenediisocyanate,hexamethylenediisothiocyanate, N,N′-ethylenebismaleinimide,N,N′-polymethylenebisiodoacetamide, 2,4-dinitrobenzenesulfonate sodiumsalt, and diazo compounds. Glutaraldehyde is preferable as across-linking reagent.

[0108] Examples of such condensation reagents include carbodiimidederivatives represented by formula RN═C═NR (or R′),N-hydroxysuccinimide, tri-n-butylamine, butyl chloroformate, andisobutyl isocyanide.

[0109] By introducing covalent layer 6 to the measuring cell to firmlymobilize physiologically active substance 4 via covalent bonds,substance 4 can be maintained immobilized when the measuring cell iswashed, which enables the cell to be used for repetitive measurementsfor another advantageous feature. The thickness of covalent layer 6 ispreferably 10 to 100 angstroms, most preferably 10 to 20 angstroms.

[0110] The physiologically active substance can also be immobilized byhydrophobic bond, by integrating substance 4 into a plasmapolymerization layer or by an additional plasma treatment.

[0111] A preferred group of the measuring chip according to the presentinvention is a measuring chip comprising a metal layer, one or moreplasma polymerization layers formed on said metal layer, and an immuneprotein or enzyme immobilized on the surface of said plasmapolymerization layer, wherein said plasma polymerization layer comprisesa monomer material selected from the group consisting of pyridine,triethylamine, diethylamine, allylamine, acrylamide, aniline,acrylonitrile, 1,2,4-triazole, 5-amino-1H-tetrazole, and acetonitrile,

[0112] Another preferred group of the measuring chip according to thepresent invention is a measuring chip comprising a metal layer, one ormore plasma polymerization layers formed on said metal layer, and animmune protein or enzyme immobilized on the surface of said plasmapolymerization layer, wherein said plasma polymerization layer comprisesa monomer material selected from the group consisting of pyridine,triethylamine, diethylamine, allylamine, acrylamide, aniline,acrylonitrile, 1,2,4-triazole, 5-amino-1H-tetrazole, and acetonitrileand wherein said immune protein or enzyme is immobilized on said plasmapolymerization layer through a cross-linking reagent or a water-solublecondensation reagent.

[0113] A cross-linking reagent for the preferred group above can beselected from the group consisting of glutaraldehyde,N-succinimidyl-4-maleimidomethylbanzoic acid,N-succinimidyl-3-maleimidobenzoic acid,N-succinimidyl-4-maleimidophenyl-4-butyric acid,N,N′-oxydimethylene-dimaleimide, N,N′-m-phenylene-dimaleimide,N,N′-p-phenylene-dimaleimide, N,N′-hexamethylene-dimaleimide,N-succinimidylmaleimidocarboxylic acid,N-succinimidyl-S-acetylmercaptoacetic acid,N-succinimidyl-3-(2-pyridyldithio)propionate, S-acetylmercaptosuccinicanhydride, methyl-3-(4′-dithiopyridyl)propionimidate,methyl-4-mercaptobutylimidate, methyl-3-mercaptopropionimidate,iminothiolene, o-carboxymethyl-hydroxylamine, azodiphenylpilmaleido,bis(sulfosuccinimidyl)sperate, 4,4′-diisothiocyano-2,2′-disulfonic acidstilbene, 4,4′-difluoro-3,3′-dinitrodiphenylsulfon,1,5-difluoro-2,4-dinitrobenzene, p-phenylenediisothiocyanate,dimethyladipimidate, dimethylpimelimidate, dimethylsuberimidate,p-azidophenacylbromide, p-azidophenylglyoxal,N-hydroxysuccinimidyl-4-azidobenzoate, 4-fluoro-3-nitrophenylazide,methyl-4-azidobenzoimidate, N-5-azido-2-nitrobenzoyloxysuccinimide,N-succinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate,1,4-benzoquinone, N-succinimidyl-3-(2′-pyridyldithio)propionate,bisdiazobenzidine, hexamethylenediisocyanate, toluenediisocyanate,hexamethylenediisothiocyanate, N,N′-ethylenebismaleinimido,N,N′-polymethylenebisiodoacetoamide, and diazo compounds; or saidcondensation reagent is one or more compounds selected from the groupconsisting of carbodiimide derivatives represented by RN═C═NR (or R′),N-hydroxysuccinimide, tri-n-butylamine, butyl chloroformate, andisobutyl isocyanide.

[0114] The measuring chip according to the present invention can beformed as follows:

[0115] First, metal thin-film 2 is formed on transparent substrate 1.Metal thin-film 2 can be formed by conventional methods such assputtering, CVD, PVD, or vacuum evaporation.

[0116] Second, plasma polymerization layer 3 is formed on metalthin-film 2. Plasmapolymerization layer 3 can be formed by plasmapolymerization using a plasma polymerization apparatus. The rate ofplasma formation is preferably 100 to 3000 angstroms/min, mostpreferably 500 to 1000 angstroms/min. If the rate exceeds 3000angstroms/min, it becomes difficult to obtain a smooth plasmapolymerization layer. More specifically, the plasma polymerization canbe preferably carried out at a monomer material flow rate of 0.05 to 100sccm at a room temperature or at a temperature of 10 to 20° C. at apressure between 1.0×10⁻² and 1.0×10² Pa using a discharge power of 20to 300 W at a discharge frequency of 10 MHz or 13.56 MHz. However,polymerization conditions are not restricted to the conditions above.

[0117] After formation of plasma polymerization layer 3, physiologicallyactive substance 4 is finally immobilized on plasma polymerization layer3. Immobilization can be done by conventional methods. For example, aspecified amount of physiologically active substance 4 can beimmobilized by contacting it with plasma polymerization layer 3 for aspecified period of time. If the measuring cell is a flow-cell type, aspecified volume of the physiologically active substance 4 can beimmobilized by contacting it with plasma polymerization layer 3 bypouring a specified volume for a specified period of time.

[0118] When an antibody is used as a physiologically active substanceand its Fab fragment is immobilized directly on plasma polymerizationlayer 3, the same treatment can be done after the antibody is partlydigested with papain. On the other hand, when the F(ab′)₂ fragment isimmobilized directly on plasma polymerization layer 3, the sametreatment can be done after the antibody is partly digested with pepsin.

[0119] When covalent bond layer 6 is formed, a cross-linking reagent ora condensation reagent is allowed to be in contact with plasmapolymerization layer 3 in the same manner as with active substance 4,after which substance 4 can be immobilized.

[0120] The measuring cell for a surface plasmon resonance sensoraccording to the present invention comprises the measuring chip. Themeasuring chip can be mounted on an optical part to be opticallyanalyzed. The term “optical part” as used herein refers to a part wherea light is projected and an evanescent wave and a surface plasmon can beinduced.

[0121] The surface plasmon resonance biosensor according to the presentinvention comprises the measuring cell.

[0122]FIG. 5 is a schematic view of one embodiment of the surfaceplasmon resonance biosensor according to the present invention. Thesurface plasmon resonance biosensor has cartridge block 7, light source8, and detector 9 and measuring chip 10 is mounted on cartridge block 7.The upper side of cartridge block 7 has a hollow and this hollow andmeasuring chip 10 construct measuring cell 71.

[0123] The body of measuring chip 10 comprises a transparent substrate,and a layer comprising a metal thin-film, a plasma polymerization layerformed under said metal film. A physiologically active substance isimmobilized on the surface of said plasma polymerization layer facingthe hollow of cartridge block 7. Measuring cell 71 is constructed fromthe hollow of cartridge block 7 and measuring chip 10; and cartridgeblock 7 has flow routes 72 and 73 providing passages to the outside ofmeasuring cell 71 and cartridge block 7, which makes measuring cell 71 aflow-cell type. However, the present invention is not restricted to thistype and a batch type cell can also be used. Using measuring cell 71 ofthis flow-cell type, a sample can be measured either continuously orintermittently. In this sensor, the sample flows into measuring cell 71via flow route 72 and is discharged after measurement via flow route 73.The flow rate of the sample is preferably 0.5 to 5 μl/min. The flow rateis controlled, for example, using a computer-operated pump.

[0124] Monochromatic light (incident light 80) is irradiated from lightsource 8 toward the optical part of measuring chip 10 and its reflectedlight 90, which is reflected by metal thin-film 2 set on the reverseside of measuring chip 10, reaches detector 9. Detector 9 can detect theintensity of reflected light 90. Light source 8 and detector 9 are notparticularly restricted, and can be any types customarily used for asurface plasmon resonance biosensor. In the sensor according to thepresent invention, the incident light is wedge-shaped and the lightreflected in different directions can be measured simultaneously.However, the present invention is not restricted to this type of sensor.The configuration of this type does not require a mobile part, therebyproducing excellent stability and durability, and enabling real timemeasurement of samples as well.

[0125] The configuration as described above yields a reflected lightintensity curve that forms a trough relative to a given angle ofincidence (see FIG. 6). The trough in the reflected light intensitycurve is due to surface plasmon resonance. Namely, when light is totallyreflected at the interface between the transparent substrate and theexterior of measuring chip 10, a surface wave known as an evanescentwave is generated at the interface and a surface wave known as a surfaceplasmon is also generated on the metal thin-film. Resonance occurs whenthe wave number of these two surface waves coincides and a part of lightenergy is consumed to excite the surface plasmon, resulting in adecrease in the intensity of the reflected light. The wave number of thesurface plasmon is affected by the refractive index of the mediumproximate to the surface of the metal thin-film. Therefore, when therefractive index of the medium changes due to an interaction between thesubstance to be measured and the physiologically active substance, asurface plasmon resonance is induced to change the angle of incidence.Thus, a change in the concentration of the substance to be measured canbe perceived by a shift of the trough in the reflected light intensitycurve. The change in the angle of incidence is called a resonance signaland a change of 10⁻⁴ degree is expressed as 1 RU. In the surface plasmonresonance biosensor of this example, highly effective and reliablemeasurement can be done if measuring chip 10 is made to be freelyattachable and detachable and disposable. Furthermore, if a covalentbond layer is provided between the plasma polymerization layer and thephysiologically active substance, measuring chip 10 can be usedrepeatedly by washing the inside of measuring cell 71, resulting in adecrease in the cost.

[0126] The surface plasmon resonance biosensor of the present inventioncan be used for quantitative or qualitative analysis, identification ofa target substance present in a sample.

EXAMPLE

[0127] The present invention is further illustrated by the followingExamples that are not intended as a limitation of the invention.

Example 1

[0128] A measuring chip having layers shown in FIG. 1 on an opticalrecognition part was constructed.

[0129] A glass plate with a thickness of 0.15 mm (18 mm×18 mm) was usedfor a transparent substrate. A chrome layer and then a gold layer weredeposited on this transparent substrate by sputtering. The sputteringwas carried out at 100 W for 40 seconds for the chrome layer and at 100W for 2 minutes and 30 seconds for the gold layer. The resulting chromelayer was 40 angstroms thick and the resulting gold layer was 500angstroms thick.

[0130] A plasma polymerization layer was formed on the metal layers. Anapparatus as shown in FIG. 7 was used for plasma polymerization.Ethanedithiol was used as a monomer material for the plasmapolymerization layer to introduce a thiol group. Conditions for plasmapolymerization were as follows:

[0131] Flow volume of monomer material: 15 sccm

[0132] Temperature: 15° C.

[0133] Pressure: 4.7 Pa

[0134] Discharge electric power: 20 W

[0135] Discharge frequency: 10 MHz, FM modulation

[0136] Duration of discharge: 60 seconds.

[0137] Under the conditions described above, a thiol group wasintroduced on the surface of plasma polymerization layer. The sensorchip with the introduced thiol group was mounted on the cartridge blockof the surface plasmon resonance biosensor and maleimidized avidin (see“Ultrahigh Sensitivity Enzyme Immunoassay” by Eiji Ishikawa) was pouredthrough a flow route into the measuring cell at a flow rate of 5 μl/minfor immobilization on the thiol group on the plasma polymerization layerfor 60 minutes. 50 μl of 10 μM-biotinized DNA were then poured and theprobe DNA was immobilized via the avidin for 10 minutes. A DNA (7.5×10⁻⁷M) having a DNA sequence complementary to this probe DNA was introducedand after the reaction, a signal of about 500 RU was obtained.Concentration of Complementary DNA (μM) 0.00075 0.0075 0.075 0.75 7.5 75RU 10 25 100 500 1000 1100

[0138] It was confirmed by an XPS analysis that the resulting membranehas a mercapto group.

[0139]FIG. 6 shows the reflected light intensity curve before and afterthe formation of the plasma polymerization layer, which show theintensity of reflected light corresponding to the angle of incidence θ).FIG. 6 shows that the plasma polymerization layer is formed on thesurface of the gold layer. The thickness of the plasma polymerizationlayer can be estimated from Δθ.

Example 2

[0140] The same apparatus and method as in Example 1 were used.

[0141] Acetonitrile was used as a monomer material for the plasmapolymerization layer. Conditions for plasma polymerization were asfollows:

[0142] Flow volume of monomer material: 1.5 sccm+Ar dilution 15 (sccm)

[0143] Temperature: room temperature

[0144] Pressure: 4.7 Pa

[0145] Discharge electric power: 80 W

[0146] Discharge frequency: 13.56 MHz

[0147] Duration of discharge: 15 seconds.

[0148] Under the conditions described above, a plasma polymerizationlayer was formed. The sensor chip was mounted on the cartridge block ofthe surface plasmon resonance biosensor, 5% glutaraldehyde was pouredthrough a flow route into the measuring cell at a flow rate of 5 μl/minfor 10 minutes and avidin (concentration: 20 μg/ml) was also poured at aflow rate of 5 μl/min to immobilize for 60 minutes. 10 μM biotin-labeledprobe RNA were then poured at a flow rate of 1 μl/min to immobilize theprobe RNA for 10 minutes. DNA (7.5×10⁻⁷ M) having a DNA sequencecomplementary to this probe RNA was introduced and after the reaction, asignal of about 500 RU was obtained. Concentration of Complementary DNA(μM) 0.00075 0.0075 0.075 0.75 7.5 75 RU 8 20 80 400 800 880

[0149] It was confirmed by the XPS analysis that the resulting membranehas a primary amine.

Example 3

[0150] The same apparatus and method as in Example 1 were used.

[0151] Conditions for plasma polymerization layer formation were thesame as in Example 2.

[0152] Under the conditions described above, a plasma polymerizationlayer was formed.

[0153] The sensor chip was mounted on the cartridge block of the surfaceplasmon resonance biosensor, 5% glutaraldehyde was poured through a flowroute into the measuring cell at a flow rate of 5 μl/min for 10 minutesand streptoavidin (concentration: 20 μg/ml) was also poured at a flowrate of 5 μl/min to immobilize for 60 minutes. 10 μM biotin-labeledprobe RNA was then poured at a flow rate of 1 μl/min for 10 minutes toimmobilize the probe RNA. DNA (7.5×10⁻⁷ M) having a DNA sequencecomplementary to this probe RNA was introduced and after the reaction, asignal of about 375 RU was obtained. Concentration of Complementary DNA(μM) 0.00075 0.0075 0.075 0.75 7.5 75 RU 7.5 18.75 75 375 750 825

[0154] It was confirmed by the XPS analysis that the resulting membranehas a primary amine.

Example 4

[0155] The same apparatus and method as in Example 1 were used.

[0156] Conditions for plasma polymerization layer formation were thesame as in Example 2 except that propargylamine was used as a monomermaterial.

[0157] Under the conditions described above, a plasma polymerizationlayer was formed. The sensor chip was mounted on the cartridge block ofthe surface plasmon resonance biosensor, 0.4 MN-ethyl-N′-(3-dimethylaminopropyl)carbodiimide was poured through a flowroute into the measuring cell at a flow rate of 5 μl/min for 10 minutesand avidin (concentration: 20 μg/ml) was also poured at a flow rate of 5μl/min to immobilize for 60 minutes. 10 μM biotin-labeled probe RNA wasthen poured at a flow rate of 1 μl/min for 10 minutes to immobilize theprobe RNA. DNA(7.5×10⁻⁷ M) having a DNA sequence complementary to thisprobe RNA was introduced and after the reaction, a signal of about 450RU was obtained. Concentration of Complementary DNA (μM) 0.00075 0.00750.075 0.75 7.5 75 RU 0.9 22.5 90 450 900 990

[0158] It was confirmed by the XPS analysis that the resulting membranehas a primary amine.

Example 5

[0159] The same apparatus and method as in Example 1 were used.

[0160] Conditions for plasma polymerization layer formation were thesame as in Example 4.

[0161] Under the conditions described above, a plasma polymerizationlayer was formed. The sensor chip was mounted on the cartridge block ofthe surface plasmon resonance biosensor, 5% glutaraldehyde was pouredthrough a flow route into the measuring cell at a flow rate of 5 μl/minfor 10 minutes and protein A (concentration: 400 μg/ml) was also pouredat a flow rate of 5 μl/min to immobilize for 60 minutes. An anti-HSAantibody (concentration: 400 μl/ml) was then poured at a flow rate of 1μl/min for 10 minutes to immobilize the antibody. An HSA antigen (10μg/ml) complementary to this anti-HSA antibody was introduced and afterthe reaction, a signal of about 250 RU was obtained. Concentration ofHSA antigen (μg/ml) 0.01 0.1 1 10 100 1000 RU 5 12.5 50 250 500 550

[0162] It was confirmed by the XPS analysis that the resulting membranehas a primary amine.

Example 6

[0163] The same apparatus and method as in Example 1 were used.

[0164] Conditions for plasma polymerization layer formation were thesame as in Example 4.

[0165] Under the conditions described above, a plasma polymerizationlayer was formed. The sensor chip was mounted on the cartridge block ofthe surface plasmon resonance biosensor, 5% glutaraldehyde was pouredthrough a flow route into the measuring cell at a flow rate of 5 μl/minfor 10 minutes and protein G (concentration: 400 μg/ml) was also pouredat a flow rate of 5 μl/min to immobilize for 60 minutes. An anti-BSAantibody (concentration: 400 μl/ml) was then poured at a flow rate of 1μl/min for 10 minutes to immobilize the antibody. A BSA antigen (10μg/ml) complementary to this anti-BSA antibody was introduced and afterthe reaction, a signal of about 225 RU was obtained. Concentration ofBSA antigen (μg/ml) 0.01 0.1 1 10 100 1000 RU 4.5 11.25 45 225 450 495

[0166] It was confirmed by the XPS analysis that the resulting membranehas a primary amine.

Example 7

[0167] The same apparatus and method as in Example 1 were used.

[0168] Conditions for plasma polymerization layer formation were thesame as in Example 4.

[0169] Under the conditions described above, a plasma polymerizationlayer was formed. The sensor chip was mounted on the cartridge block ofthe surface plasmon resonance biosensor, 5% glutaraldehyde was pouredthrough a flow route into the measuring cell at a flow rate of 5 μl/minfor 10 minutes and mannose-binding lectin (concentration: 200 μg/ml) wasalso poured at a flow rate of 5 μl /min to immobilize for 60 minutes.

[0170] A sugar (10 μg/ml) complementary to this mannose-binding lectinwas introduced and after the reaction, a signal of about 200 RU wasobtained. Concentration of sugar (μg/ml) 0.01 0.1 1 10 100 1000 RU 4 1040 200 400 440

[0171] It was confirmed by the XPS analysis that the resulting membranehas a primary amine.

Example 8

[0172] The same apparatus and method as in Example 1 were used.

[0173] Conditions for plasma polymerization layer formation were thesame as in Example 4 except that pyridine was used as a monomermaterial.

[0174] Under the conditions described above, a plasma polymerizationlayer was formed. The sensor chip was mounted on the cartridge block ofthe surface plasmon resonance biosensor, 5% glutaraldehyde was pouredthrough a flow route into the measuring cell at a flow rate of 5 μl/minfor 10 minutes and an anti-BSA antibody (concentration: 400 μg/ml) wasalso poured at a flow rate of 5 μl/min to immobilize for 60 minutes. ABSA antigen (10 μg/ml) complementary to this anti-BSA antibody wasintroduced and after the reaction, a signal of about 187.5 RU wasobtained. Concentration of BSA antigen (μg/ml) 0.01 0.1 1 10 100 1000 RU3.75 9.375 37.5 187.5 375 412.5

[0175] It was confirmed by the XPS analysis that the resulting membranehas a primary amine.

Example 9

[0176] The same apparatus and method as in Example 1 were used.

[0177] Conditions for plasma polymerization layer formation were thesame as in Example 8 except that acrylonitrile was used as a monomermaterial.

[0178] Under the conditions described above, a plasma polymerizationlayer was formed. The sensor chip was mounted on the cartridge block ofthe surface plasmon resonance biosensor, 5% glutaraldehyde was pouredthrough a flow route into the measuring cell at a flow rate of 5 μl/minfor 10 minutes and an anti-BSA antibody (concentration: 400 μg/ml) wasalso poured at a flow rate of 5 μl/min to immobilize for 60 minutes. ABSA antigen (10 μg/ml) complementary to this anti-BSA antibody wasintroduced and after the reaction, a signal of about 200 RU wasobtained. Concentration of BSA antigen (μg/ml) 0.01 0.1 1 10 100 1000 RU4 10 40 200 400 440

[0179] It was confirmed by the XPS analysis that the resulting membranehas a primary amine.

Example 10

[0180] The same apparatus and method as in Example 1 were used.

[0181] Conditions for plasma polymerization layer formation were thesame as in Example 9 except that ethanethiol was used as a monomermaterial.

[0182] Under the conditions described above, a plasma polymerizationlayer was formed. The sensor chip was mounted on the cartridge block ofthe surface plasmon resonance biosensor and maleimidized anti-BSAantibody was poured through a flow route at a flow rate of 5 μl/min toimmobilize for 60 minutes. A BSA antigen (10 μg/ml) complementary tothis anti-BSA antibody was introduced and after the reaction, a signalof about 200 RU was obtained. Concentration of BSA antigen (μg/ml) 0.010.1 1 10 100 1000 RU 4 10 40 200 400 440

[0183] It was confirmed by the XPS analysis that the resulting membranehas a mercapto group.

Example 11

[0184] The same apparatus and method as in Example 1 were used.

[0185] Conditions for plasma polymerization layer formation were thesame as in Example 10 except that thiophene was used as a monomermaterial.

[0186] Under the conditions described above, a plasma polymerizationlayer was formed. The sensor chip was mounted on the cartridge block ofthe surface plasmon resonance biosensor and maleimidized anti-BSAantibody was poured through a flow route at a flow rate of 5 μl/min toimmobilize for 60 minutes. A BSA antigen (10 μg/ml) complementary tothis anti-BSA antibody was introduced and after the reaction, a signalof about 187.5 RU was obtained. Concentration of BSA antigen (μg/ml)0.01 0.1 1 10 100 1000 RU 3.75 9.375 37.5 187.5 375 412.5

[0187] It was confirmed by the XPS analysis that the resulting membranehas a mercapto group.

Example 12

[0188] The same apparatus and method as in Example 1 were used.

[0189] Conditions for plasma polymerization layer formation were thesame as in Example 11 except that acetonitrile was used as a monomermaterial.

[0190] Under the conditions described above, a plasma polymerizationlayer was formed. The sensor chip was mounted on the cartridge block ofthe surface plasmon resonance biosensor, 5% glutaraldehyde was pouredthrough a flow route into the measuring cell at a flow rate of 5 μl/minfor 10 minutes and an anti-HSA antibody (concentration: 400 μg/ml) wasalso poured at a flow rate of 5 μl/min to immobilize for 60 minutes. HSAantigen (10 μg/ml) complementary to this anti-HSA antibody wasintroduced and after the reaction, a signal of about 250 RU wasobtained. Concentration of HSA antigen (μg/ml) 0.01 0.1 1 10 100 1000 RU5 10 50 250 500 550

[0191] It was confirmed by the XPS analysis that the resulting membranehas a primary amine.

Example 13

[0192] The same apparatus and method as in Example 1 were used.

[0193] Conditions for plasma polymerization layer formation were thesame as in Example 12.

[0194] Under the conditions described above, a plasma polymerizationlayer was formed. The sensor chip was mounted on the cartridge block ofthe surface plasmon resonance biosensor, 5% glutaraldehyde was pouredthrough a flow route into the measuring cell at a flow rate of 5 μl/minfor 10 minutes and the Fab fragment of an anti-HSA antibody(concentration: 400 μg/ml) was also poured at a flow rate of 5 μl/min toimmobilize for 60 minutes.

[0195] A HSA antigen (10 μg/ml) complementary to this Fab fragment ofthe anti-HSA antibody was introduced and after the reaction, a signal ofabout 275 RU was obtained. Concentration of HSA antigen (μg/ml) 0.01 0.11 10 100 1000 RU 5.5 11 55 275 550 605

[0196] It was confirmed by the XPS analysis that the resulting membranehas a primary amine.

Example 14

[0197] The same apparatus and method as in Example 1 were used.

[0198] Conditions for plasma polymerization layer formation were thesame as in Example 13.

[0199] Under the conditions described above, a plasma polymerizationlayer was formed. The sensor chip was mounted on the cartridge block ofthe surface plasmon resonance biosensor, 5% glutaraldehyde was pouredthrough a flow route into the measuring cell at a flow rate of 5 μl/minfor 10 minutes and the F(ab)₂ fragment of an anti-HSA antibody(concentration: 400 μg/ml) was also poured at a flow rate of 5 μl/min toimmobilize for 60 minutes.

[0200] A HSA antigen (10 μg/ml) complementary to this F(ab)₂ fragment ofthe anti-HSA antibody was introduced and after the reaction, a signal ofabout 300 RU was obtained. Concentration of HSA antigen (μg/ml) 0.01 0.11 10 100 1000 RU 6 12 60 300 600 660

[0201] It was confirmed by the XPS analysis that the resulting membranehas a primary amine.

Example 15

[0202] The same apparatus and method as in Example 1 were used.

[0203] Conditions for plasma polymerization layer formation were asfollows:

[0204] (1) Monomer: Hexadiene

[0205] Flow volume of monomer material: 1.5 sccm+Ar dilution 15 (sccm)

[0206] Temperature: room temperature

[0207] Pressure: 1.6 Pa

[0208] Discharge electric power: 80 W

[0209] Discharge frequency: 13.56 MHz

[0210] Duration of discharge: 15 seconds;

[0211] (2) Monomer: Ethylenediamine

[0212] Flow volume of monomer material: 1.5 sccm

[0213] Temperature: room temperature

[0214] Pressure: 1.6 Pa

[0215] Discharge electric power: 80 W

[0216] Discharge frequency: 13.56 MHz

[0217] Duration of discharge: 5 seconds.

[0218] The targeted surface was obtained by the two-step process above.

[0219] Under the conditions described above, a plasma polymerizationlayer was formed. The sensor chip was mounted on the cartridge block ofthe surface plasmon resonance biosensor, 5% glutaraldehyde was pouredthrough a flow route into the measuring cell at a flow rate of 5 μl/minfor 10 minutes and an anti-HSA antibody (concentration: 400 μg/ml) wasalso poured at a flow rate of 5 μl/min to immobilize for 60 minutes. AHSA antigen (10 μg/ml) complementary to this anti-HSA antibody wasintroduced and after the reaction, a signal of about 250 RU wasobtained. Concentration of HSA antigen (μg/ml) 0.01 0.1 1 10 100 1000 RU5 10 50 250 500 550

[0220] It was confirmed by the XPS analysis that the resulting membranehas a primary amine.

Example 16

[0221] The same apparatus and method as in Example 1 were used.

[0222] Conditions for plasma polymerization layer formation were asfollows:

[0223] (1) Monomer: Hexamethyldisiloxane

[0224] Flow volume of monomer material: 1.5 sccm+Ar dilution 15 (sccm)

[0225] Temperature: room temperature

[0226] Pressure: 1.6 Pa

[0227] Discharge electric power: 80 W

[0228] Discharge frequency: 13.56 MHz

[0229] Duration of discharge: 15 seconds;

[0230] (2) Monomer: Ethylenediamine

[0231] Flow volume of monomer material: 1.5 sccm

[0232] Temperature: room temperature

[0233] Pressure: 1.6 Pa

[0234] Discharge electric power: 80 W

[0235] Discharge frequency: 13.56 MHz

[0236] Duration of discharge: 5 seconds.

[0237] The targeted surface was obtained by the two-step process above.

[0238] Under the conditions described above, a plasma polymerizationlayer was formed. The sensor chip was mounted on the cartridge block ofthe surface plasmon resonance biosensor, 5% glutaraldehyde was pouredthrough a flow route into the measuring cell at a flow rate of 5 μl/minfor 10 minutes and an anti-HSA antibody (concentration: 400 μg/ml) wasalso poured at a flow rate of 5 μl/min to immobilize for 60 minutes. AHSA antigen (10 μg/ml) complementary to this anti-HSA antibody wasintroduced and after the reaction, a signal of about 225RU was obtained.Concentration of HSA antigen (μg/ml) 0.01 0.1 1 10 100 1000 RU 4.5 9 45225 450 495

[0239] It was confirmed by the XPS analysis that the resulting membranehas a primary amine.

Example 17

[0240] The same apparatus and method as in Example 1 were used.

[0241] Conditions for plasma polymerization layer formation were thesame as in Example 2 except that propylamine was used as a monomermaterial.

[0242] Under the conditions described above, a plasma polymerizationlayer was formed. The sensor chip was mounted on the cartridge block ofthe surface plasmon resonance biosensor, 5% glutaraldehyde was pouredthrough a flow route into the measuring cell at a flow rate of 5 μl/minfor 10 minutes and avidin (concentration: 20 μg/ml) was also poured at aflow rate of 5 μl/min to immobilize for 60 minutes. 10 μM biotin-labeledprobe RNA was poured at a flow rate of 1 μl/min to immobilize the probeRNA for 10 minutes. DNA (7.5×10⁻⁷ M) having a DNA sequence complementaryto this probe RNA was introduced and after the reaction, a signal ofabout 400 RU was obtained. Concentration of Complementary DNA (μM)0.00075 0.0075 0.075 0.75 7.5 75 RU 8 20 80 400 800 880

[0243] It was confirmed by the XPS analysis that the resulting membranehas a primary amine.

Example 18

[0244] The same apparatus and method as in Example 1 were used.

[0245] Conditions for plasma polymerization layer formation were asfollows:

[0246] (1) Monomer: Propargyl Alcohol

[0247] Flow volume of monomer material: 1.5 sccm

[0248] Temperature: room temperature

[0249] Pressure: 1.6 Pa

[0250] Discharge electric power: 20 W

[0251] Discharge frequency: 13.56 MHz

[0252] Duration of discharge: 15 seconds;

[0253] (2) Monomer: Oxygen (Plasma Treatment)

[0254] Flow volume of monomer material: 1.5 sccm

[0255] Temperature: room temperature

[0256] Pressure: 1.6 Pa

[0257] Discharge electric power: 20 W

[0258] Discharge frequency: 13.56 MHz

[0259] Duration of discharge: 5 seconds.

[0260] The targeted surface was obtained by the two-step process above.

[0261] Under the conditions described above, a plasma polymerizationlayer was formed. The sensor chip was mounted on the cartridge block ofthe surface plasmon resonance biosensor, a 0.5 M carbodiimide solutionwas poured through a flow route into the measuring cell at a flow rateof 5 μl/min for 10 minutes and an anti-HSA antibody (concentration: 400μg/ml) was also poured at a flow rate of 5 μl/min to immobilize for 60minutes. A HSA antigen (10 μg/ml) complementary to this anti-HSAantibody was introduced and after the reaction, a signal of about 250 RUwas obtained. Concentration of HSA antigen (μg/ml) 0.01 0.1 1 10 1001000 RU 5 10 50 250 500 550

[0262] It was confirmed by the XPS analysis that the resulting membranehas a carboxyl group.

Example 19

[0263] The same apparatus and method as in Example 1 were used.

[0264] Conditions for plasma polymerization layer formation were thesame as in Example 2 except that propagylamine was used as a monomermaterial.

[0265] Under the conditions described above, a plasma polymerizationlayer was formed. The sensor chip was mounted on the cartridge block ofthe surface plasmon resonance biosensor, 0.5 M carbodiimide was pouredthrough a flow route into the measuring cell at a flow rate of 5 μl/minfor 10 minutes and behenic acid (concentration: 400 μg/ml) was alsopoured at a flow rate of 5 μl/min to immobilize for 60 minutes. Skatole(10 μg/ml) complementary to this behenic acid was introduced and afterthe reaction, a signal of about 225 RU was obtained. Concentration ofskatole (μg/ml) 0.01 0.1 1 10 100 1000 RU 4.5 9 45 225 450 495

[0266] It was confirmed by the XPS analysis that the resulting membranehas a primary amine.

Example 20

[0267] Example 20 shows a formation of hydrophobic bond.

[0268] A layer comprising chrome and gold was formed on a transparentsubstrate (glass plate) by sputtering. A plasma polymerization layer inwhich trifluoroethylene was used as a monomer was then formed on theresulting metal layer under the following conditions:

[0269] Flow volume: 1.5 sccm

[0270] Temperature: room temperature

[0271] Pressure: 5 Pa

[0272] Discharge electric power: 50 W

[0273] Discharge frequency: 13.56 MHz

[0274] Duration of discharge: 30 seconds.

[0275] The plasma polymerization layer obtained under the conditionsdescribed above was hydrophobic. An anti-HSA antibody (concentration:100 μg/ml) was allowed to flow at a flow rate of 5 μl/min for 60 minutesto immobilize the antibody via hydrophobic bond. HSA at a specifiedconcentration was further reacted with this antibody-immobilized plasmapolymerization layer. The following results were obtained. Concentrationof HSA antigen (μg/ml) 0.01 0.1 1 10 100 1000 RU 3 6 30 150 300 330

Example 21

[0276] Example 21 shows an inclusion of an antibody by plasmapolymerization.

[0277] A layer comprising chrome and gold was formed on a transparentsubstrate (glass plate) by sputtering. A plasma polymerization layer inwhich propargyl alcohol was used as a monomer was then formed on theresulting metal layer under the following conditions:

[0278] Flow volume: 1.5 sccm

[0279] Temperature: room temperature

[0280] Pressure: 1.6 Pa

[0281] Discharge electric power: 20 W

[0282] Discharge frequency: 13.56 MHz

[0283] Duration of discharge: 15 seconds.

[0284] The plasma polymerization layer obtained under the conditionsdescribed above was highly hydrophilic. An antibody solution(concentration: 100 μg/ml) was spread evenly on this propargyl alcoholplasma polymerization layer and after drying, plasma treatment wasfurther carried out on this surface under the following conditions:

[0285] Flow volume: 1.5 sccm

[0286] Temperature: room temperature

[0287] Pressure: 1.6 Pa

[0288] Discharge electric power: 20 W

[0289] Discharge frequency: 13.56 MHz

[0290] Duration of discharge: 8 seconds.

[0291] HSA at a specified concentration was reacted with the membrane inwhich the antibody was thus integrated and immobilized by plasmatreatment. The following results were obtained, from which a calibrationcurve could be drawn. Concentration of 0.01 0.1 1 10 100 1000 HSAantigen (μg/ml) RU 5 10 50 250 500 550

1. A measuring chip for a surface plasmon resonance sensor comprising a metal layer and one or more plasma polymerization layers formed on said metal layer.
 2. A measuring chip for a surface plasmon resonance sensor comprising a metal layer, one or more plasma polymerization layers formed on said metal layer, and a nucleic acid immobilized on the surface of said plasma polymerization layer.
 3. A measuring chip for a surface plasmon resonance sensor according to claim 2, wherein said nucleic acid is DNA, RNA or PNA.
 4. A measuring chip for a surface plasmon resonance sensor comprising a metal layer, one or more plasma polymerization layers formed on said metal layer, and a non-immune protein immobilized on the surface of said plasma polymerization layer.
 5. A measuring chip for a surface plasmon resonance sensor according to claim 4, wherein said non-immune protein is either avidin, streptoavidin, biotin or a receptor.
 6. A measuring chip for a surface plasmon resonance sensor comprising a metal layer, one or more plasma polymerization layers formed on said metal layer, and an immunoglobulin-binding protein immobilized on the surface of said plasma polymerization layer.
 7. A measuring chip for a surface plasmon resonance sensor according to claim 6, wherein said immunoglobulin-binding protein is protein A, protein G, or a rheumatoid factor.
 8. A measuring chip for a surface plasmon resonance sensor comprising a metal layer, one or more plasma polymerization layers formed on said metal layer, and a sugar-binding protein immobilized on the surface of said plasma polymerization layer.
 9. A measuring chip for a surface plasmon resonance sensor according to claim 8, wherein said sugar-binding protein is lectin.
 10. A measuring chip for a surface plasmon resonance sensor comprising a metal layer, one or more plasma polymerization layers formed on said metal layer, and a sugar-recognizing sugar chain immobilized on the surface of said plasma polymerization layer.
 11. A measuring chip for a surface plasmon resonance sensor comprising a metal layer, one or more plasma polymerization layers formed on said metal layer, and a fatty acid or a fatty acid ester immobilized on the surface of said plasma polymerization layer.
 12. A measuring chip for a surface plasmon resonance sensor according to claim 11, wherein said fatty acid or fatty acid ester is either stearic acid, alachidic acid, behenic acid, ethyl stearate, ethyl arachidate, or ethyl behanate.
 13. A measuring chip for a surface plasmon resonance sensor comprising a metal layer, one or more plasma polymerization layers formed on said metal layer, and a polypeptide or oligopeptide having ligand-binding activity immobilized on the surface of said plasma polymerization layer.
 14. A measuring chip for a surface plasmon resonance sensor according to claim 13, wherein said polypeptide or oligopeptide is produced by using genetic engineering techniques or chemical synthesis methods.
 15. A measuring chip according to any one of claims 1 to 14, which further comprises an optically transparent substrate on which said metal layer is formed.
 16. A measuring chip according to any one of claims 1 to 15, wherein said plasma polymerization layer comprises a compound having one or more groups selected from the group consisting of —COOH, —CHO, —SH, —NH₂, —OH, ═NH, —CONH₂, —NCO, —CH═CH₂, ═C═O, and


17. A measuring chip according to any one of claims 1 to 15, wherein said plasma polymerization layer consists of two or more layers.
 18. A measuring chip according to any one of claims 1 to 15, wherein said monomer material of a plasma polymerization layer is a compound containing nitrogen.
 19. A measuring chip according to claim 18, wherein said compound containing nitrogen is CH₃—(CH₂)_(n)—NH₂ (wherein n is an integer from 1 to 6) and/or NH₂—(CH₂)_(n)—NH₂ (wherein n is an integer from 1 to 6).
 20. A measuring chip according to claim 18, wherein said compound containing nitrogen is selected from the group consisting of pyridine, ethylenediamine, hexamethylenediamine, n-propylamine, monoethylamine, triethylamine, diethylamine, allylamine, acrylamide, aniline, acrylonitrile, 1,2,4-triazole, 5-amino-1H-tetrazole, propargylamine and acetonitrile.
 21. A measuring chip according to any one of claims 1 to 15, wherein a monomer material of said plasma polymerization layer is a compound containing sulfur.
 22. A measuring chip according to claim 21, wherein said compound containing sulfur is selected from the group consisting of dimethyl sulfide, methyl disulfide, ethanethiol, ethanedithiol, thiophen, mercaptoethanol and dithreitol.
 23. A measuring chip according to any one of claims 1 to 15, wherein said monomer material of a plasma polymerization layer is a compound containing a halogen.
 24. A measuring chip according to any one of claims 1 to 15, wherein said monomer material of a plasma polymerization layer comprises a compound having one or more groups selected from the group consisting of —COOH, —CHO, —SH, —NH₂, —OH, ═NH, —CONH₂, —NCO, —CH═CH₂, ═C═O and


25. A measuring chip according to any one of claims 1 to 15, wherein said monomer material of a plasma polymerization layer is a compound having —C≡CCH₂OH.
 26. A measuring chip according to any one of claims 1 to 15, wherein said monomer material of a plasma polymerization layer is a carbohydrate compound comprising C and H.
 27. A measuring chip according to any one of claims 1 to 15, wherein said monomer material of a plasma polymerization layer is an organic metal compound.
 28. A measuring chip according to claim 27, wherein said organic metal compound is an organic silicon compound.
 29. A measuring chip according to claim 28, wherein said organic silicon compound is selected from the group consisting of tetramethylsilane, tetramethyldisiloxane, hexamethyldisiloxane, hexamethyldisilazane, hexamethylcyclotrisilazane, dimethylaminotrimethylsilane, trimethylvinylsilane, tetramethoxysilane, aminopropyltriethoxysilane, octadecyldiethoxymethylsilane, hexamethyldisilane, and divinyltetramethyldisiloxane.
 30. A measuring chip according to any one of claims 1 to 15, wherein plasma treatment is applied to said plasma polymerization layer with a polymeric or non-polymeric monomer.
 31. A measuring chip according to claim 30 wherein said non-polymeric monomer material is selected from the group consisting of nitrogen, ammonium, hydrazine, hydrogensulfide, hydrogendisulfide, oxygen, hydrogen, water, halogen gas, and rare gas.
 32. A measuring chip according to any one of claims 1 to 15, wherein said monomer material of plasma polymerization layer is a mixture of two or more of the substances claimed in claims 18 to
 31. 33. A measuring chip for a surface plasmon resonance sensor comprising a metal layer, one or more plasma polymerization layers formed on said metal layer, and an immune protein or enzyme immobilized on the surface of said plasma polymerization layer, wherein said plasma polymerization layer comprises a monomer material selected from the group consisting of pyridine, triethylamine, diethylamine, allylamine, acrylamide, aniline, acrylonitrile, 1,2,4-triazole, 5-amino-1H-tetrazole, and acetonitrile.
 34. A measuring chip for a surface plasmon resonance sensor comprising a metal layer, one or more plasma polymerization layers formed on said metal layer, and an immune protein or enzyme immobilized on the surface of said plasma polymerization layer, wherein said plasma polymerization layer comprises a monomer material selected from the group consisting of the compounds claimed in any one of claims 21 to
 32. 35. A measuring chip according to claim 33 or 34, wherein said immune protein is an antibody.
 36. A measuring chip according to claim 33 or 34, wherein said immune protein is a Fab fragment of an antibody.
 37. A measuring chip according to claim 33 or 34, wherein said immune protein is a F(ab)2 fragment of an antibody.
 38. A measuring chip according to claim 2 or 3, wherein said nucleic acid is immobilized on said plasma polymerization layer through a cross-linking reagent or a condensation reagent.
 39. A measuring chip according to claim 4 or 5, wherein said non-immune protein is immobilized on said plasma polymerization layer through a cross-linking reagent or a condensation reagent.
 40. A measuring chip according to claim 6 or 7, wherein said immunoglobulin-binding protein is immobilized on said plasma polymerization layer through a cross-linking reagent or a condensation reagent.
 41. A measuring chip according to claim 8 or 9, wherein said sugar-binding protein is immobilized on said plasma polymerization layer through a cross-linking reagent or a condensation reagent.
 42. A measuring chip according to claim 10, wherein said sugar-recognizing sugar chain is immobilized on said plasma polymerization layer through a cross-linking reagent or a condensation reagent.
 43. A measuring chip according to claim 11 or 12, wherein said fatty acid or fatty acid ester is immobilized on said plasma polymerization layer through a cross-linking reagent or a condensation reagent.
 44. A measuring chip according to claim 13, wherein said polypeptide or oligopeptide is immobilized on said plasma polymerization layer through a cross-linking reagent or a condensation reagent.
 45. A measuring chip according to any one of claims 38 to 44, wherein said cross-linking reagent is one or more compounds selected from the group consisting of glutaraldehyde, periodic acid, N-succinimidyl-2-maleimidoacetic acid, N-succinimidyl-4-maleimidobutyric acid, N-succinimidyl-6-maleimidohexanic acid, N-succinimidyl-4-maleimidomethylcyclohexan-1-carboxylic acid, N-sulfosuccinimidyl-4-maleimidomethylcyclohexane-1-carboxylic acid, N-succinimidyl-4-maleimidomethylbanzoic acid, N-succinimidyl-3-maleimidobenzoic acid, N-sulfosuccinimidyl-3-maleimidobenzoic acid, N-succinimidyl-4-maleimidophenyl-4-butyric acid, N-sulfosuccinimidyl-4-maleimidophenyl-4-butyric acid, N,N′-oxydimethylene-dimaleimide, N,N′-o-phenylene-dimaleimide, N,N′-m-phenylene-dimaleimide, N,N′-p-phenylene-dimaleimide, N,N′-hexamethylene-dimaleimide, N-succinimidylmaleimidocarboxylic acid, N-succinimidyl-S-acetylmercaptoacetic acid, N-succinimidyl-3-(2-pyridyldithio)propionate, S-acetylmercaptosuccinic anhydride, methyl-3-(4′-dithiopyridyl)propionimidate, methyl-4-mercaptobutylimidate, methyl-3-mercaptopropionimidate, iminothiolene, o-carboxymethyl-hydroxylamine, azodiphenylpilmaleido, bis(sulfosuccinimidyl)sperate, 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene, 4,4′-difluoro-3,3′-dinitrodiphenylsulfon, 1,5-difluoro-2,4-dinitrobenzene, p-phenylenediisothiocyanate, dimethyladipimidate, dimethylpimelimidate, dimethylsuberimidate, p-azidophenacylbromide, p-azidophenylglyoxal, N-hydroxysuccinimidyl-4-azidobenzoate, 4-fluoro-3-nitrophenylazide, methyl-4-azidobenzoimidate, N-5-azido-2-nitrobenzoyloxysuccinimide, N-succinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate, 1,4-benzoquinone, N-succinimidyl-3-(2′-pyridyldithio)propionate, N-(4-maleimidobutyloxy)sulfosuccinimide sodium salt, N-(6-maleimidocaproyloxy)sulfosuccinimide sodium salt, N-(8-maleimidocaproyloxy)sulfosuccinimide sodium salt, N-(11-maleimidoundecanoyloxy)sulfosuccinimide sodium salt, N-[2-(1-piperazinyl)ethyl]maleimide bichloric acid, bisdiazobenzidine, hexamethylenediisocyanate, toluenediisocyanate, hexamethylenediisothiocyanate, N,N′-ethylenebismaleinimide, N,N′-polymethylenebisiodoacetamide, 2,4-dinitrobenzenesulfonate sodium salt, and diazo compounds; or said condensation reagent is one or more compounds selected from the group consisting of carbodiimide derivatives represented by RN═C═NR (or R′), N-hydroxysuccinimide, tri-n-butylamine, butyl chloroformate, and isobutyl isocyanide.
 46. A measuring chip according to any one of claims 33 to 37, wherein said immune protein or enzyme is immobilized on said plasma polymerization layer through a cross-linking reagent or a water-soluble condensation reagent.
 47. A measuring chip according to claim 46, wherein said cross-linking reagent is one or more compounds selected from the group consisting of glutaraldehyde, N-succinimidyl-4-maleimidomethylbanzoic acid, N-succinimidyl-3-maleimidobenzoic acid, N-succinimidyl-4-maleimidophenyl-4-butyric acid, N,N′-oxydimethylene-dimaleimide, N,N′-m-phenylene-dimaleimide, N,N′-p-phenylene-dimaleimide, N,N′-hexamethylene-dimaleimide, N-succinimidylmaleimidocarboxylic acid, N-succinimidyl-S-acetylmercaptoacetic acid, N-succinimidyl-3-(2-pyridyldithio)propionate, S-acetylmercaptosuccinic anhydride, methyl-3-(4′-dithiopyridyl)propionimidate, methyl-4-mercaptobutylimidate, methyl-3-mercaptopropionimidate, iminothiolene, o-carboxymethyl-hydroxylamine, azodiphenylpilmaleido, bis(sulfosuccinimidyl)sperate, 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene, 4,4′-difluoro-3,3′-dinitrodiphenylsulfon, 1,5-difluoro-2,4-dinitrobenzene, p-phenylenediisothiocyanate, dimethyladipimidate, dimethylpimelimidate, dimethylsuberimidate, p-azidophenacylbromide, p-azidophenylglyoxal, N-hydroxysuccinimidyl-4-azidobenzoate, 4-fluoro-3-nitrophenylazide, methyl-4-azidobenzoimidate, N-5-azido-2-nitrobenzoyloxysuccinimide, N-succinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate, 1,4-benzoquinone, N-succinimidyl-3-(2′-pyridyldithio)propionate, bisdiazobenzidine, hexamethylenediisocyanate, toluenediisocyanate, hexamethylenediisothiocyanate, N,N′-ethylenebismaleinimido, N,N′-polymethylenebisiodoacetoamide, and diazo compounds; or said condensation reagent is one or more compounds selected from the group consisting of carbodiimide derivatives represented by RN═C═NR (or R′), N-hydroxysuccinimide, tri-n-butylamine, butyl chloroformate, and isobutyl isocyanide.
 48. A measuring chip for a surface plasmon resonance sensor comprising a metal layer, a plasma polymerization layer formed on said metal layer, a substance immobilized on the surface of said plasma polymerization layer, and an additional plasma polymerization layer or plasma-treated layer formed on said plasma polymerization layer.
 49. A measuring chip according to claim 48, wherein said substance to be immobilized is selected from the group consisting of a nucleic acid, a non-immune protein, an immunoglobulin-binding protein, a sugar-binding protein, a sugar-recognizing sugar chain, a fatty acid or a fatty acid ester, a polypeptide or oligopeptide having ligand-binding activity, an immune protein, and an enzyme.
 50. A measuring chip for a surface plasmon resonance sensor, comprising a metal layer, one or more plasma polymerization layers formed on said metal layer, and a substance immobilized on said plasma polymerization layer through a hydrophobic bond.
 51. A measuring chip according to claim 50, wherein said substance to be immobilized is selected from the group consisting of a nucleic acid, a non-immune protein, an immunoglobulin-binding protein, a sugar-binding protein, a sugar-recognizing sugar chain, a fatty acid or a fatty acid ester, a polypeptide or oligopeptide having ligand-binding activity, an immune protein, and an enzyme.
 52. A method for producing a measuring chip for a surface plasmon resonance sensor comprising the steps of forming a metal layer on an optically transparent substrate, forming one or more plasma polymerization layers on said metal layer, and then immobilizing a nucleic acid on the surface of said plasma polymerization layer.
 53. A method for producing a measuring chip for a surface plasmon resonance sensor comprising the steps of forming a metal layer on an optically transparent substrate, forming one or more plasma polymerization layers on said metal layer, and then immobilizing a non-immune protein on the surface of said plasma polymerization layer.
 54. A method for producing a measuring chip for a surface plasmon resonance sensor comprising the steps of forming a metal layer on an optically transparent substrate, forming one or more plasma polymerization layers on said metal layer, and then immobilizing an immunoglobulin-binding protein on the surface of said plasma polymerization layer.
 55. A method for producing a measuring chip for a surface plasmon resonance sensor comprising the steps of forming a metal layer on an optically transparent substrate, forming one or more plasma polymerization layers on said metal layer, and then immobilizing a sugar-binding protein on the surface of said plasma polymerization layer.
 56. A method for producing a measuring chip for a surface plasmon resonance sensor comprising the steps of forming a metal layer on an optically transparent substrate, forming one or more plasma polymerization layers on said metal layer, and then immobilizing a sugar-recognizing sugar chain on the surface of said plasma polymerization layer.
 57. A method for producing a measuring chip for a surface plasmon resonance sensor comprising the steps of forming a metal layer on an optically transparent substrate, forming one or more plasma polymerization layers on said metal layer, and then immobilizing a fatty acid or fatty acid ester on the surface of said plasma polymerization layer.
 58. A method for producing a measuring chip for a surface plasmon resonance sensor comprising the steps of forming a metal layer on an optically transparent substrate, forming one or more plasma polymerization layers on said metal layer, and then immobilizing a polypeptide or oligopeptide having a ligand binding capability on the surface of said plasma polymerization layer.
 59. A method according to any one of claims 52 to 58, wherein the plasma polymerization layer is formed by a plasma-treatment using a monomer material claimed in any one of claims 18 to
 32. 60. A method for producing a measuring chip for a surface plasmon resonance sensor comprising the steps of forming a metal layer on an optically transparent substrate, forming one or more plasma polymerization layers on said metal layer by a plasma-treatment using a monomer material claimed in any one of claims 18 to 32, and then immobilizing an immune protein or enzyme on the surface of said plasma polymerization layer.
 61. A measuring cell for a surface plasmon resonance sensor comprising a measuring chip according to any one of claims 1 to
 51. 62. A measuring cell according to claim 61, wherein said chip is optically analyzed.
 63. A surface plasmon resonance biosensor comprising a measuring chip according to any one of claims 1 to 51 or a measuring cell according to claim 61 or
 62. 